Project Summary/Abstract Our laboratory, and now many others, have added well over 100 noncanonical amino acids (ncAAs) to the genetic codes of single and multi-cellular organisms [1, 2]. These amino acids include spectroscopic probes, metal ion chelators, post-translationally modified amino acids and stable analogues thereof, amino acids with bio-orthogonal chemical reactivity, photocrosslinkers, and a host of other novel functions. Moreover, we have distributed this technology to hundreds of laboratories throughout the world to probe protein structure and function both inside and outside the cell, to engineer proteins with enhanced catalytic, physical and biological properties, and to create novel biotherapeutics. Here we propose to further expand our ability to manipulate the genetic code and apply this approach to important problems in biomedical research including exploring the function of short open reading frame encoded peptides, evolving proteins with enhanced properties, and genetically encoding ncAAs in hematopoietic stem cells and lineages derived therefrom in mice.